Dynamics of the peptidogylcan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium

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1 SUPPLEMENTAL MATERIAL Dynamics of the peptidogylcan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium Emöke Cserti, Sabine Rosskopf, Yi-Wei Chang, Sabrina Eisheuer, Lars Selter, Jian Shi, Christina Regh, Ulrich Koert, Grant J. Jensen, Martin Thanbichler

2 SUPPLEMENTAL FIGURES Figure S1. Morphological changes of H. neptunium over the course of multiple cell cycles. Swarmer cells were immobilized in a microfluidic flow cell and followed by time-lapse DIC microscopy at 15-min intervals. The lengths and widths of the (mother) cell bodies were measured immediately after immobilization (swarmer cell), right before the onset of bud formation (stalked cell), at the end of the first cell cycle immediately before the release of the mature bud (1 st division), and at the end of the fourth and eighth cell cycle. The data are shown as box plots, with the bar indicating the median, the box the interquartile range, and the whiskers the 5 th and 95 th percentile. Significant differences are indicated by asterisks (***; t-test, p < 10-8 ). The lack of significance (t-test; p > 0.05) is indicated by ns. n = 31. 1

3 Figure S2. Morphology of PG hydrolase mutants. The H. neptunium wild type and strains EC36 (ΔlmdA), EC53 (ΔlmdB), EC38 (ΔlmdD), EC39 (ΔlmdE), EC90 (ΔlmdF), SR11 (ΔdacB), SR08 (ΔdacH), EC46 (ΔdacL), EC95 (ΔmltA), SR07 (ΔgplA), SR33 (ΔrlpA), EC21 (ΔsltA), SR20 (ΔmltB), and SR18 (ΔamiC) were grown to exponential phase and analyzed by DIC microscopy. Scale bar: 3 µm. (B) Cell lengths of the hydrolase mutant strains. The lengths of cells grown as described in (A) were quantified and shown th th as box plots, with the bar representing the median, the boxes the interquartile range, and the whiskers the 5 and 95 percentile. None of the distributions obtained for the mutant strains is significantly different from that of the wild-type strain (ttest, p > 0.01). 2

4 Figure S3. Characterization of strains producing fluorescently tagged PG biosynthetic proteins. (A) Cell length analysis. The SW H. neptunium wild type and strains SR28 (dacl-mcherry), SR14 (venus-pbp2), SE161 (venus- pbp3), EC63 (mreb-mcherry ), and EC93 (yfp-rodz) were grown to exponential phase and imaged by DIC microscopy. The lengths of the cells were measured and th th shown in box plots, with the bar representing the median, the boxes the interquartile range, and the whiskers the 5 and 95 percentile. None of the distributions is significantly different from that of the wild-type strain (t-test, p > 0.01). (B) Stability of the fusion proteins analyzed in this study. The H. neptunium wild type and strains SR28 (dacl-mcherry), EC70 (Pcu::Pcu-amiCSW mcherry), EC63 (mreb-mcherry ), EC93 (yfp-rodz), SE161 (venus-pbp3), and SR14 (venus-pbp2) were grown to exponential phase and subjected to Western blot analysis with an anti-mcherry or anti-gfp antibody (performed as described by Jung et al., 2015). Strain EC70 was induced for 24 h with 300 µm CuSO4 before analysis. Arrows indicate the full-length fusion proteins. 3

5 Figure S4. Growth rates of peptidoglycan hydrolase mutants. The growth of the H. neptunium wild type and the indicated PG hydrolase-deficient strains EC36 (ΔlmdA), EC53 (ΔlmdB), EC38 (ΔlmdD), EC39 (ΔlmdE), EC90 (ΔlmdF), SR11 (ΔdacB), SR08 (ΔdacH), EC46 (ΔdacL), EC95 (ΔmltA), SR20 (ΔmltB), SR33 (ΔrlpA), EC21 (ΔsltA), SR07 (ΔgplA), and SR18 (ΔamiC) was followed using a microplate reader. The doubling times were normalized to the value obtained for the wild-type strain (3.6 ± 0.3 h). Figure S5. (A) Morphology of PG synthase mutants. Cells of strains SR31 (ΔldtA), CR04 (ΔldtB), SR32 (ΔldtAB), and EC89 (ΔmtgA) were grown to exponential phase and analyzed by DIC microscopy. Scale bar: 3 µm. (B) Growth rates of PG synthase mutants. The growth of the H. neptunium wild type and the indicated PG synthase-deficient strains EC26 (Δpbp1X), EC27 (Δpbp1C), and EC57 (Δpbp1X Δpbp1C), SR31 (ΔldtA), CR04 (ΔldtB), SR32 (ΔldtAB), and EC89 (ΔmtgA) was followed using a microplate reader. The doubling times were normalized to the value obtained for the wild-type strain (3.6 ± 0.3 h). 4

6 Figure S6. Subcellular distribution of Venus-PBP3. Shown are demographs of swarmer and stalked cells of strain SE161 (venuspbp3). Figure S7. Structural model of H. neptunium MreB. Structural model of H. neptunium MreB (MreBHNE; in blue), generated using the crystal structure of ADP-bound MreB from C. crescentus (MreBCC; PDB 4CZF; in orange) as a template. 5

7 Figure S8. Effect of the MreB inhibitor A22 on MreB. (A) Inhibition of H. neptunium growth by the MreB inhibitor A22. Exponentially growing H. neptunium wild-type cells were diluted into fresh media containing the indicated concentrations of A22. Subsequently, their growth was monitored for 30 h using a microplate reader. (B) Effect of A22 on cell morphology. Cells were grown to exponential phase and exposed for 24 h to 90 µm A22. After washing and resuspension in medium lacking the inhibitor, cells were cultivated further to monitor the gradual recovery of wild-type morphology. Samples were taken at the indicated time points after addition (+) or removal (-) of A22 and analyzed by DIC microscopy. Scale bar: 3 µm. (C,D) Quantitative analysis of changes in cell shape after A22 treatment. Cells were grown to exponential phase and exposed to the indicated concentrations of A22 for 24 h. After imaging by DIC microscopy, the lengths (C) and widths (D) of their cell bodies (excluding stalks and buds) were measured. The data are shown as box plots, as described in Figure S3A. Distributions significantly different from that of the control culture (0 µm) are indicated (***; t-test, p < 10 in C and p < 10 in D). n = 100 SW for each concentration. (E) Delocalization of MreB-mCherry in the presence of A22 or MP265. Cells of strain EC63 (mrebsw mcherry ) were grown to exponential phase, exposed to A22 (35 µm) or MP265 (250 µm) for 1 h, and subjected to fluorescence microscopy. After determination of the fluorescence profiles of random subpopulations of cells, the data were plotted as demographs. Cells grown in the absence of any inhibitor are analyzed as a control. 6

8 Figure S9. Delocalization of PG incorporation after inhibition of MreB. Cells of the H. neptunium wild-type strain were grown for 5 h in the absence (control) or presence of 250 µm MP265, labeled with HADA, and analyzed by fluorescence microscopy. The demographs show fluorescence profiles obtained from random subpopulations of cells from each condition. SUPPLEMENTAL MOVIES Movie S1. Time-lapse analysis of growing H. neptunium cells. Wild-type cells were grown in a microfluidic device and imaged at 15 min intervals by DIC microscopy. Scale bar: 2 µm. 7

9 SUPPLEMENTAL TABLES Table S1. Proteins investigated in this work. Protein ORF number Predicted function Lytic enzymes Metallopeptidases LmdA HNE_0632 Peptidase (M23 family) LmdB HNE_0633 Peptidase (M23 family) LmdC HNE_2628 Peptidase (M23 family) LmdD HNE_2982 Peptidase (M23 family) LmdE HNE_3210 Peptidase (M23 family) LmdF HNE_3409 Peptidase (M23 family) Carboxypeptidases DacB HNE_0402 D-Ala-D-Ala carboxy-/endopeptidase DacH HNE_1025 D-Ala-D-Ala carboxypeptidase DacL HNE_1814 D-Ala-D-Ala carboxypeptidase Glycosyl hydrolases GplA HNE_0445 Glycosyl hydrolase family protein MltA HNE_0008 Lytic murein transglycosylase MltA MltB HNE_3349 Lytic murein transglycosylase RlpA HNE_1815 Lytic transglycosylase SltA HNE_2801 Lytic transglycosylase, SLT family Amidase AmiC HNE_0674 N-acetylmuramoyl-L-Ala amidase Synthetic enzymes Bifunctional PBPs PBP1A HNE_1911 Penicillin-binding protein 1A PBP1C HNE_3002 Penicillin-binding protein 1C PBP1X HNE_0768 Penicillin-binding protein, 1A family Monofunctional PBPs PBP2 HNE_2934 Penicillin-binding protein 2 PBP3 HNE_3030 Penicillin-binding protein 3, FtsI L,D-Transpeptidases LdtA HNE_0929 L,D-transpeptidase LdtB HNE_3551 L,D-transpeptidase Synthetic Transglycosylase MtgA HNE_3102 Monosynthetic transglycosylase Regulatory factors MreB HNE_2937 Actin homologue MreB RodZ HNE_0620 MreB-associated protein RodZ 8

10 Table S2. H. neptunium strains used in this study. Strains Genotype/description Construction References LE670 Wild type (ATCC 15444) Leifson (1964) CR04 LE670 ΔHNE3551 (ldtb) In-frame deletion of HNE3551 in LE670 using pcr04 This study EC21 LE670 ΔHNE2801 (slta) In-frame deletion of HNE2801 in LE670 using pec28 This study EC26 LE670 ΔHNE0768 (pbp1x) In-frame deletion of HNE0768 in LE670 using pec22 This study EC27 LE670 ΔHNE3002 (pbp1c) In-frame deletion of HNE3002 in LE670 using pec26 This study EC36 LE670 ΔHNE0632 (lmda) In-frame deletion of HNE0632 in LE670 using pec34 This study EC38 LE670 ΔHNE2982 (lmdd) In-frame deletion of HNE2982 in LE670 using pec38 This study EC39 LE670 ΔHNE3210 (lmde) In-frame deletion of HNE3210 in LE670 using pec39 This study EC46 LE670 ΔHNE1814 (dacl) In-frame deletion of HNE1814 in LE670 using pec64 This study EC53 LE670 ΔHNE0633 (lmdb) In-frame deletion of HNE0633 in LE670 using pec35 This study EC57 LE670 ΔHNE0768 (pbp1x) In-frame deletion of HNE3002 in EC26 using pec26 This study ΔHNE3002 (pbp1c) EC63 LE670 mreb-mcherry SW Gene replacement in LE670 using pec87 This study EC70 LE670 P cu::p cu-hne0674 (amic)- Integration of pec115 in LE670 This study mcherry EC89 LE670 ΔHNE3102 (mtga) In-frame deletion of HNE3102 in LE670 using pec65 This study EC90 LE670 ΔHNE3409 (lmdf) In-frame deletion of HNE3409 in LE670 using pec126 This study EC93 LE670 yfp-hne0620 (rodz) Gene replacement in LE670 using pec129 This study EC95 LE670 ΔHNE0008 (mlta) In-frame deletion of HNE0008 in LE670 using pec157 This study SE161 LE670 venus-hne3030 (pbp3) Gene replacement in LE670 using pse68 This study SR07 LE670 ΔHNE0445 (glpa) In-frame deletion of HNE0445 in LE670 using psr02 This study SR08 LE670 ΔHNE1025 (dach) In-frame deletion of HNE1025 in LE670 using psr03 This study SR11 LE670 ΔHNE0402 (dacb) In-frame deletion of HNE0402 in LE670 using psr01 This study SR14 LE670 venus-hne2934 (pbp2) Gene replacement in LE670 using psr06 This study SR18 LE670 ΔHNE0674 (amic) In-frame deletion of HNE0674 in LE670 using psr22 This study SR20 LE670 ΔHNE3349 (mltb) In-frame deletion of HNE3349 in LE670 using psr19 This study SR24 LE670 HNE0633 (lmdb)- mcherry Gene replacement in LE670 using psr13 This study SR26 LE670 HNE3409 (lmdf)-mcherry Gene replacement in LE670 using psr17 This study SR28 LE670 HNE1814 (dacl)-mcherry Gene replacement in LE670 using psr38 This study SR31 LE670 ΔHNE0929 (ldta) In-frame deletion of HNE0929 in LE670 using pec174 This study SR32 LE670 ΔHNE0929 (ldta) In-frame deletion of HNE0929 in CR04 using pec174 This study ΔHNE3551 (ldtb) SR33 LE670 ΔHNE1815 (rlpa) In-frame deletion of HNE1815 in LE670 using pec172 This study Table S3. E. coli strains used in this study. Strains Genotype/description References TOP10 F mcra Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 reca1 arad139 Δ(ara leu) 7697 galu galk Invitrogen rpsl (Str R ) enda1 nupg WM6034 thrb1004 pro thi rpsl hsds laczδm15 RP Δ(araBAD)567 ΔdapA1341::[erm pir(wt)] W. Metcalf (unpublished) Table S4. General plasmids used in this work. Jung et al., 2014 Plasmids Description References pcchyc-2 Integrating plasmid to construct C-terminal fusions to mcherry under the control of P cu, Kan R pchyc-2 Integrating plasmid to construct C-terminal fusions to mcherry at a site of interest, Kan R Thanbichler et al., 2007 pcvenc-2 Integrating plasmid to construct C-terminal fusions to Venus under the control of P cu, Kan R Jung et al., 2014 pcvenn-3 Integrating plasmid to construct N-terminal fusions to Venus under the control of P cu, Rif R Jung et al., 2014 pcyfpc-2 Integrating plasmid to construct C-terminal fusions to YFP under the control of P cu, Kan R Jung et al., 2014 pcyfpn-2 Integrating plasmid to construct N-terminal fusions to YFP under the control of P cu, Kan R Jung et al., 2014 pnpts138 sacb-containing suizide vector used for double homologous recombination, Kan R M.R. Alley, unpublished pzvenn-2 Integrating plasmid to construct N-terminal fusions to Venus under the control of P zn, Kan R Jung et al.,

11 Table S5. Plasmids generated in this work. Plasmids Description pcr04 pec115 pec12 pec126 pec129 pec157 pec172 pec174 pec22 pec26 pec28 pec34 HNE3551 (ldtb) pcchyc-2 bearing HNE0674 (amic)- mcherry pcchyc-2 bearing HNE3409 (lmdf)- mcherry HNE3409 (lmdf) replacing rodz with yfprodz (HNE0620) HNE0008 (mlta) HNE1815 (rlpa) HNE0929 (ldta) HNE0768 (pbp1x) HNE3002 (pbp1c) HNE2801 (slta) HNE0632 (lmda) Construction a) amplification of the HNE3551 flanking regions from LE670 chrom. DNA using primers ocr16+ocr17 (upstream) and ocr18+ocr19 (downstream), b) restriction of the upstream fragment with HindIII and BamHI, restriction of the downstream fragment with BamHI and NheI c) triple ligation with pnpts138 cut with PstI and NheI a) amplification of HNE_0674 from LE670 chrom. DNA using primers oec157+ss271, restriction with NdeI and KnpI, b) ligation with pcchyc-2 cut with NdeI and KnpI a) amplification of HNE3409 from LE670 chrom. DNA using primers oec23+oec24, restriction with KpnI and NdeI b) ligation with pcchyc-2 cut with KpnI and NdeI a) amplification of the HNE3409 flanking regions from LE670 chrom. DNA using primers oec289+oec290 (upstream) and oec291+oec292 (downstream), b) restriction of the upstream fragment with HindIII and BamHI, restriction of the downstream fragment with BamHI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of three fragments for extension overlap PCR using primers oec244+oec245 (template: LE670 chrom. DNA), oec246+oec247 (template: pcyfpc-2), oec248+oec249 (template: LE670 chrom. DNA) b) fusion of the three fragments by extension-overlap PCR to generate upstream-eyfp- HNE0620 using primers oec244+oec249 c) restriction of upstream-eyfp-hne0620 with HindIII and NheI and ligation with pnpts138 cut with HindIII and NheI a) amplification of the HNE0008 flanking regions from LE670 chrom. DNA using primers oec313+oec314 (upstream) and oec315+oec316 (downstream), b) restriction of the upstream fragment with HindIII and BamHI, restriction of the downstream fragment with BamHI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of the HNE1815 flanking regions from LE670 chrom. DNA using primers oec326+oec327 (upstream) b) amplification of the downstream region from LE670 chrom. DNA by a nested PCR using first primers oec326+oe331 and then primers oec328+oec329 c) restriction of the upstream flank with BamHI and HindIII, restriction of downstream flank with BamHI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of HNE0929 flanking regions from LE670 chrom. DNA using primers oec338+oec339 (upstream) and oec340+oec341 (downstream) b) restriction of upstream flank with BamHI and KpnI, restriction of downstream flank with KpnI and NheI c) triple ligation with pnpts138 digested with BamHI and NheI a) amplification of HNE0768 flanking regions from LE670 chrom. DNA using primers oec33+oec34 (upstream) and oec35+oec36 (downstream), b) restriction of upstream fragment with PstI and HindIII, restriction of the downstream fragment with HindIII and NheI c) triple ligation with pnpts138 cut with PstI and NheI a) amplification of the HNE3002 flanking regions from LE670 chrom. DNA using primers oec49+oec50 (upstream) and oec51+oec52 (downstream), b) restriction of the upstream fragment with EcoRI and HindIII, restriction of the downstream fragment with HindIII and NheI c) triple ligation with pnpts138 cut with EcoRI and NheI a) amplification of the HNE2801 flanking regions from LE670 chrom. DNA using primers oec55+oec56 (upstream) and oec57+oec58 (downstream) b) restriction of the upstream fragment with HindIII and NheI, restriction of downstream fragment with NheI and EcoRI c) triple ligation with pnpts138 cut with HindIII and EcoRI a) amplification of the HNE0632 flanking regions from LE670 chrom. DNA using primers oec84+oec85 (upstream) and oec86+oec87 (downstream) b) restriction of the upstream fragment with BamHI and HindIII, restriction of the downstream fragment with BamHI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI 10

12 Table S5. Plasmids generated in this work (continued). pec35 pec38 pec39 pec48 pec55 pec64 pec65 pec7 pec87 pse68 psr01 psr02 psr03 HNE0633 (lmdb) HNE2982 (lmdd) HNE3210 (lmde) pcchyn-2 bearing mcherry-mreb pcvenn-3 bearing venus-hne2934 (pbp2) HNE1814 (dacl) HNE3102 (mtga) pcchyc-2 bearing HNE1814 (dacl)- mcherry replacing mreb with mreb-mcherry SW pnpts138 derivative for replacing native ftsi with venus-ftsi (HNE3030) HNE0402 (dacb) HNE0445 (gpla) HNE1025 (dach) a) amplification of the HNE0663 flanking regions from LE670 chrom. DNA using primers oec88+oec89 (upstream) and oec90+oec91 (downstream), b) restriction of the upstream fragment with PstI and HindIII, restriction of the downstream fragment with HindIII and NheI c) triple ligation with pnpts138 cut with PstI and NheI a) amplification of the HNE2982 flanking regions from LE670 chrom. DNA using primers oec92+oec93 (upstream) and oec94+oec95 (downstream) b) restriction of the upstream fragment with PstI and HindIII, restriction of the downstream fragment with HindIII and NheI c) triple ligation with pnpts138 cut with PstI and NheI a) amplification of the HNE3210 flanking regions from LE670 chrom. DNA using primers oec96+oec97 (upstream) and oec98+oec99 (downstream), b) restriction of the upstream fragment with EcoRI and HindIII, restriction of the downstream fragment with HindIII and NheI c) triple ligation with pnpts138 cut with EcoRI and NheI a) amplification of HNE_2937 from LE670 chrom. DNA using primers oec123+oec124, restriction with KpnI and NheI b) ligation with pcchyn-2 cut with KpnI and NheI a) amplification of HNE2934 from LE670 chrom. DNA using primers oec9+oec10, restriction with KpnI and NheI b) ligation into pcchyn-2 cut with KpnI and NheI a) amplification of HNE1814 flanking regions from LE670 chrom. DNA using primers oec137+oec138 (upstream) and oec139+oec140 (downstream) b) restriction of upstream flankwith EcoRI and PstI, restriction of downstream flank with EcoRI and NheI c) triple ligation with pnpts138 cut PstI and NheI a) amplification of the HNE3102 flanking regions from LE670 chrom. DNA using primers oec295+oec142 (upstream) and oec143+oec296 (downstream), b) restriction of the upstream fragment with HindIII and EcoRI, restriction of the downstream fragment with EcoRI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of HNE1814 from LE670 chrom. DNA using primers oec27 and oec28, restriction with KpnI and NdeI b) ligation into pcchyc-2 cut with KpnI and NdeI a) amplification of three fragments for extension overlap PCR using primers oec182+oec184 (template pec48), oec183+oec186 (template pcchyc-2), oec185+oec124 (template pec48) b) fusion of the fragments by extension-overlap PCR to generate an mreb -mcherry- mreb fragment using primers oec182+oec124 c) digestion of mreb -mcherry- mreb with HindIII and NheI and ligation into pnpts138 cut with HindIII and NheI a) amplification of the upstream region of HNE3030 from ATCC15444 chrom. DNA using primers ose88+ose89 b) amplification of HNE3030 from LE670 chrom. DNA using primers ose20+ose21, restriction with KpnI and SacI, ligation into pzvenn-2 cut with KpnI and SacI, amplification of venus-ftsi' from the resulting plasmid using primers ose90+ose91 c) fusion of the two PCR-products by overlap-extension PCR d) digestion with HindIII and BamHI and ligation into pnpts138 cut with HindIII BamHI a) amplification of the HNE0402 flanking regions from LE670 chrom. DNA using primers osr01+osr02 (upstream) and osr03+osr04 (downstream) b) restriction of the upstream fragment with HindIII and EcoRI, restriction of the downstream fragment with EcoRI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of the HNE0445 flanking regions from LE670 chrom. DNA using primers osr05+osr06 (upstream) and osr07+osr08 (downstream) b) restriction of the upstream fragment with HindIII and EcoRI, restriction of the downstream fragment with EcoRI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI a) amplification of the HNE1025 flanking regions from LE670 chrom. DNA using primers osr9+osr10 (upstream) and osr11+osr12 (downstream) b) restriction of the upstream fragment with HindIII and EcoRI, restriction of the downstream fragment with EcoRI and NheI c) triple ligation with pnpts138 cut with HindIII and NheI 11

13 Table S5. Plasmids generated in this work (continued). psr06 psr17 psr19 psr22 psr38 pnpts138 derivative for replacing native HNE2934 with venus- HNE2934 (pbp2) pnpts138 derivative for replacing native HNE3409 with HNE3409 (lmdf)-mcherry HNE3349 (mltb) HNE0674 (amic) pnpts138 derivative for replacing native HNE1814 with HNE1814 (dacl)-mcherry a) amplification of two fragments for extension overlap PCR using primers osr32+osr31 (template: pec55) and oec48+oec33 (template: LE670 chrom. DNA) b) fusion of both fragments by extension-overlap PCR to generate upstream-venus-pbp2 using oec48+oec31 c) restriction of upstream-venus-hne2934 with HindIII and NheI and ligation with pnpts138 digested with HindIII and NheI a) amplification of two fragments for extension overlap PCR using primers osr70+osr72 (template: pec12) and osr71+oec103 (template: LE670 chrom. DNA) b) fusion of both fragments by extension overlap PCR to generate HNE3409-mCherrydownstream using primers osr70+oec103 c) restriction of HNE3409-mCherry-downstream with HindIII and NheI and ligation with pnpts138 digested with HindIII and NheI a) amplification of two fragments for extension overlap PCR using primers osr81+osr84 (template: pcvenc-2) and osr82+osr83 (template LE670 chrom. DNA) b) fusion of both fragments by extension overlap PCR to generate HNE3349-mCherrydownstream using primers osr81+osr82 c) digestion of HNE3349-mCherry-downstream with HindIII and NheI and ligation with pnpts138 cut with HindIII and NheI a) amplification of the HNE0674 flanking regions from LE670 chrom. DNA using primers osr89+osr90 (upstream) and osr91+osr92 (downstream) b) restriction of the upstream fragment with HindIII and BamHI, restriction of the downstream fragment with BamHI and NheI c) triple ligation with pnpts138 digested with HindIII and NheI a) amplification of two fragments for extension overlap PCR using primers osr73+osr76 (template: pec07) and osr74+osr75 (template: LE670 chrom. DNA) b) fusion of both fragments by extension overlap PCR to generate HNE1814-mCherrydownstream using primers os73+osr74 c) restriction of HNE1814-mCherry-downstream with HindIII and NheI and ligation with pnpts138 cut with HindIII and NheI 12

14 Table S6. Oligonucleotides used in this work. ID Oligonucleotide Sequence 1 Restriction site ocr16 HNE_3551_Del1f TTTTAAGCTTGTATGGGTTGTGACGGCCGCG HindIII ocr17 HNE_3551_Del1r CTAGGATCCCAGTTTTCCCTCTGAAAAGGC BamHI ocr18 HNE_3551_Del2f GAGGGATCCCGTCTGACGGGCCGTTCGGCG BamHI ocr19 HNE_3551_Del2r TATAGCTAGCGCTTTCTCGCTCATCAAGATG NheI oec9 HNE_2934_for AAAAGGTACCATGAGCCGGAAAAGTAAAAACGCTG KpnI oec10 HNE_2934_rev TTTTGCTAGCTCATGCCGGGTCACCGGTGTTG NheI oec23 HNE_3409_for AAACATATGCTGAAAAGACGCTTATCCGCC NdeI oec24 HNE_3409_rev TTTGGTACCTTCGATGATCTCGTAGCCTTCGGG KpnI oec27 HNE_1814_for TTTCATATGGTGTTTGCGGCCCTACTGACCCTCG NdeI oec28 HNE_1814_rev ATAGGTACCGTCATTGCTGCTGGAAATAAACGTG KpnI oec33 HNE_0768_del1 TATCTGCAGCACGAAGCCCGGCATGTCCTCAT PstI oec34 HNE_0768_del2 TATAAGCTTGGGTGAAGGCACCCGCTGGTATA HindIII oec35 HNE_0768_del3 ATATAAGCTTCAGGCGGAATAGGCCAAAGAACAG HindIII oec36 HNE_0768_del4 ATATGCTAGCGCTATGCGTGGCGATGGCGGACCT NheI oec49 HNE_3002_del1 AAAGAATTCGGATCAATGCCGCGAATGAAGTGGG EcoRI oec50 HNE_3002_del2 TTTAAGCTTGACCCCGCCTGACGCCCGGTCTG HindIII oec51 HNE_3002_del3 TTTTAAGCTTGCGTTTCGGCACGGACTGGCCC HindIII oec52 HNE_3002_del4 TTTTGCTAGCGCGCTTCCTCGGCATGGACGGC NheI oec55 HNE_2801_del1 ATATAAGCTTTATAAAGCACCACGGGCAGGGCGAC HindIII oec56 HNE_2801_del2 ATATGCTAGCAATCCGGAGCATGGTTCGGCT NheI oec57 HNE_2801_del3 ATATGCTAGCGGCCGCCGCTGACCGGAAGTGTC NheI oec58 HNE_2801_del4 ATATGAATTCGTAAGCACGCCAATGGTCGCCGA EcoRI oec84 HNE_0632_del1 TATAAGCTTGGTGTCCGAGCAGGCCCGCGAGCAT HindIII oec85 HNE_0632_del2 ATAGGATCCAACTCCGCCCGGATGGCAAACGCCC BamHI oec86 HNE_0632_del3 ATAGGATCCGACAGGAATCCGAGGCAGCTTTCGC BamHI oec87 HNE_0632_del4 TTTTGCTAGCGACACCGCCTATGCCCACCTCTCGC NheI oec _del1new TATCTGCAGGGCGCAGGCGCTGGCCCCGCGTGGG PstI oec89 HNE_0633_del2 TATGAATTCCCGCGTGAAGCAACGCCCGTAAGCC EcoRI oec90 HNE_0633_del3 TATGAATTCGGTTGCCGGGGCGATGGGAAGGTCG EcoRI oec91 HNE_0633_del4 TTTTGCTAGCCTTCCTGGGCCTCTGCGGGCACATC NheI oec92 HNE_2982_del1 TATCTGCAGTCTATCAGGAAGACGGCAAGGTTTG PstI oec93 HNE_2982_del2 TATAAGCTTCCCTATGAAGGGGCGTTGCAGGCCG HindIII oec94 HNE_2982_del3 TATAAGCTTGGCCGCGAGCGCAAGAGCAATCGCC HindIII oec95 HNE_2982_del4 TTTTGCTAGCCATTCTTCCCAAGCCCGGCATTGAC NheI oec96 HNE_3210_del1 TATGAATTCGGCCGTTGATCTCGGTGATATAGTC EcoRI oec97 HNE_3210_del2 TATAAGCTTCTTAATCCTGCAGAGTGGTTGTCGC HindIII oec98 HNE_3210_del3 TATAAGCTTAGGGCGGAAAAAGCGCATGCGCGGA HindIII oec99 HNE_3210_del4 TTTTGCTAGCGGGCGTTGGCGTTGGGTGGCGCTTG NheI oec103 HNE_3409_del4 TTTTGCTAGCTCGGTTGCGGGCGACAGCGCCGTTG NheI oec _for TTTGGTACCATGATCGGTAGCCTCCTCGGCCTG KpnI oec _rev TTTTGCTAGCTCAGACTTCCGGGCAGAGGACGC NheI oec137 HNE_1814_del1 TATCTGCAGGCTTTTGGTCTGCGCTCGAACGTG PstI oec138 HNE_1814_del2 ATATGAATTCGTTCGCACGTTTATTTCCAGCAGC EcoRI oec139 HNE_1814_del3 ATATGAATTCGAGCGCTCCGAGGGTCAGTAGGGC EcoRI oec140 HNE_1814_del4 TATAGCTAGCCGTGTCGGCGCCGGTTGCTGC NheI oec142 HNE_3102_del2 ATATGAATTCGTAGGCGGGGGCGGAGGAGTTCATG EcoRI oec143 HNE_3102_del3 ATATGAATTCCCACCGGCCCCACAGAACTCCGCC EcoRI oec157 HNE_0674_for TATACATATGGGCACTCAACGCCCGTCTCATAC NdeI oec _for2 TATAAGCTTATGATCGGTAGCCTCCTCGGCCTG HindIII oec SF-eol-for CCGCGATGCCGCCTGAAAGCGGATCGAGCATGGTGAGCAAGGGCGAGGAG - oec SF-eol-rev CTCCTCGCCCTTGCTCACCATGCTCGATCCGCTTTCAGGCGGCATCGCGG - oec SF-eol-for2 GGACGAGCTGTACAAGTCTGGTGCACCGGGTAACGGCACCGGCATGGCGC - oec SF-eol-rev2 GCGCCATGCCGGTGCCGTTACCCGGTGCACCAGACTTGTACAGCTCGTCC - oec244 HNE_0620_eol_for TATAGCTAGCCGCGCTCGACCATAAAGGT NheI oec245 HNE_0620_eol_rev TCCTCGCCCTTGCTCACCATATTCTACCAGTCACTTCGAC - oec246 HNE_0620_eol_for2 GTCGAAGTGACTGGTAGAATATGGTGAGCAAGGGCGAGGA - oec247 HNE_0620_eol_rev2 TGGGTCATGTTGTGTGCCATATGCATATTAATTAAGGCGC - oec248 HNE_0620_eol_for3 GCGCCTTAATTAATATGCATATGGCACACAACATGACCCA - oec249 HNE_0620_eol_rev3 TTTAAGCTTCGGCCAGTGTGCGGCTGAG HindIII oec289 HNE_3409_del1new TTTAAGCTTCCCGGCCAGAAGGACACAAAATGAG HindIII oec290 HNE_3409_del2new ATAGGATCCGGATAGTCCGATGGCGGATAAGCGT BamHI oec291 HNE_3409_del3new ATAGGATCCTGGGTCATCCCCGAAGGCTACGAGA BamHI 13

15 Table S6. Oligonucleotides used in this work (continued). oec292 HNE_3409_del4new TATAGCTAGCCGCGCTGTATATGCCGCCGGC NheI oec295 HNE_3102_del1new TTTAAGCTTGGGCCGCCACACAAACCTCGTCAGC HindIII oec296 HNE_3102_del4new TATAGCTAGCGCAGGCGCTCATCGAGGCTGACC NheI oec313 HNE_0008_del1 TTTAAGCTTAGGATTTCGCCGCCAACATGTC HindIII oec314 HNE_0008_del2 TTTGGATCCAAGGGCGGAAAACAGGAGAGCA BamHI oec315 HNE_0008_del3 TTTGGATCCGTCGCCGCGCGCACGCCTGCGCC BamHI oec316 HNE_0008_del4 TATAGCTAGCCAAGGACACGGCAGACGAGTAC NheI oec326 HNE_1815_del1 TTTAAGCTTGAACAGTATGCGATGGAAAAT HindIII oec327 HNE_1815_del2 TTTGGATCCCCGGCCAACAAAAGTCGAACG BamHI oec328 HNE_1815_del3 TTTGGATCCGGCACGGCGGACGGCCGGATC BamHI oec329 HNE_1815_del4 TATAGCTAGCCGTCATCTTGGCGAGCCGGGC NheI oec _del_check_rev GAAGATTCCAGATGCCCCGTC - oec338 HNE_0929_del1 TTTGGATCCCGCAAGCGTCAGCAGCAACAG BamHI oec339 HNE_0929_del2 CGCGGTACCAAAGAGCAGAAGAAACAGGGT KpnI oec340 HNE_0929_del3 CGCGGTACCGTCCCGGAAGGAACCTTCCTG KpnI oec341 HNE_0929_del4 TATAGCTAGCCCGGCCTGCATATCCTCGTTG NheI ose20 FtsI-Fwd ATGGTACCATGAGCGAAGCCTCCCGGGATC KpnI ose21 FtsI-Rev ATGAGCTCTCAAAGTTCGCTCCTGTTCTGGG SacI ose88 FtsIflankingup-fwd AATTAAAAGCTTGCAGAGCGCATATTGAAGCCCGGC HindIII ose89 FtsIflankingup-rev CTTGCTCACCATCTGGGCGCCTCCGCAGG - ose90 VenusftsI'-fwd GGCGCCCAGATGGTGAGCAAGGGCGAGGAGCTG - ose91 VenusftsI'-rev ATGGATCCAGGCGTATCGCTGGCGCGGTTGG BamHI osr01 HNE_0402_del1 TAGTAAGCTTGCGATCGAGACGGATGGATTT HindIII osr02 HNE_0402_del2 TATGAATTCCGCGCCAGAGAGAAAGCGGGG EcoRI osr03 HNE_0402_del3 TATGAATTCGCCAATCTGGTGCGCATCGCC EcoRI osr04 HNE_0402_del4 TATAGCTAGCTATCTCCGCTTCAGGGTTTCC NheI osr05 HNE_0445_del1 TAGTAAGCTTAACCTGCAGAACAACTAGACG HindIII osr06 HNE_0445_del2 TATGAATTCCAGCATGAACAGACGGCGCAC EcoRI osr07 HNE_0445_del3 TATGAATTCGTCGATCTCAACCGGGCAGCA EcoRI osr08 HNE_0445_del4 TATAGCTAGCGGCCATCCATGACTGACAGGT NheI osr09 HNE_1025_del1 TAGTAAGCTTTCCAGCAGCACATCAGCGTAG HindIII osr10 HNE_1025_del2 TATGAATTCATGGACTACACCCGCCGTCAG EcoRI osr11 HNE_1025_del3 TATGAATTCGCGAAACGCACGAGACCAGACT EcoRI osr12 HNE_1025_del4 TATAGCTAGCGCGTAGCCCGACTGGATCTGG NheI osr31 HNE_2934_int_rev2 TAGTAAGCTTACCGCGTCGAGGCCATCTGCA HindIII osr32 HNE_2934_int_for3 GAGCCATCAGGGCAGACGACATGGTGAGCAAGGGCGAGGA - osr33 HNE_2934_int_rev4 TCCTCGCCCTTGCTCACCATGTCGTCTGCCCTGATGGCTC - osr70 HNE_3409_int_for1 TAGTAAGCTTCTCGCCCTTCTGCCCGGTAAA HindIII osr71 HNE_3409_int_for3 TGGACGAGCTGTACAAGTAAAACAAAAAGGGCGGCTCTCG - osr72 HNE_3409_int_rev4 CGAGAGCCGCCCTTTTTGTTTTACTTGTACAGCTCGTCCA - osr73 HNE_1814_int_for1 TAGTAAGCTTCGTCGGCTGTGCGCGATGGCGTGC HindIII osr74 HNE_1814_int_rev2 TATAGCTAGCTGGTCTGCGCTCGAACGTGTC NheI osr75 HNE_1814_int_for3 TGGACGAGCTGTACAAGTAAGAGGGTGTTCGCACGTTTAT - osr76 HNE_1814_int_rev4 ATAAACGTGCGAACACCCTCTTACTTGTACAGCTCGTCCA - osr81 HNE_3349_int_for1 TAGTAAGCTTCCGCAGGGGTTTGACTTCAGC HindIII osr82 HNE_3349_int_rev2 TATAGCTAGCCCCTTCCTGGTCTATGCTGTG NheI osr83 HNE_3349_int_for3 TGGACGAGCTGTACAAGTAAGCGCGTATCCGGGGCGGCGC - osr84 HNE_3349_int_rev4 GCGCCGCCCCGGATACGCGCTTACTTGTACAGCTCGTCCA - osr89 HNE_0674_del1 TAGTAAGCTTCCTCTTCAGCCGCTTCCTTCA HindIII osr90 HNE_0674_del2 AAGGATCCCAGCTCGGCGGTATGAGACGG BamHI osr91 HNE_0674_del3 AAGGATCCTGCTCGAACTTGGCTTCCTGA BamHI osr92 HNE_0674_del4 TATAGCTAGCAAAGGGTGTGGCCGCCGTCAG NheI oss271 HNE_0674-rev ATGGTACCTTGGGACGCGAGGCGGAGATCCTG KpnI 1 Restriction sites are indicated in boldface. 14

16 SUPPLEMENTAL REFERENCES Jung, A., Eisheuer, S., Cserti, E., Leicht, O., Strobel, W., Möll, A., Schlimpert, S., Kühn, J., and Thanbichler, M. (2015) Molecular toolbox for genetic manipulation of the stalked budding bacterium Hyphomonas neptunium. Appl Environ Microbiol 81: Thanbichler, M., Iniesta, A. A., and Shapiro, L. (2007) A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res 35: e